2A used in the Creation of Transgenic Plants, Microbes & Animals.

The first types of genetically modified organisms created using 2A to co-express multiple proteins were plants. Initially as a research tool, but also to introduce multigenic 'traits' - e.g. to improve the hardiness (drought-resistance; Kwon et al., 2004) or nutritional qualities (Ralley et al., 2004) of plants.

Transgenic animals have also been created: in the case of mice the coexpression of the multiple (6 different) chains required to create a functional T-cell receptor complex (Szymczak et al., 2004). Transgenic mice have also been created co-expressing lucifease and fluorescent proteins to study the transplantation of bone marrow cells (Cao et al., 2005) using whole-body imaging techniques. Transgenic mice co-expressing different fluorescent proteins targeted to different sub-cellular sites have produced superb images of these proteins in a wide range of tissues (e.g Trichas et al., 2008).

This technology has also been used to great effect in exciting recent work involving the generation of other transgenic animal species e.g. drosophila, zebrafish and pigs.

References citing the use of 2A;

Halpin, C., Cooke, S.E., Barakate, A., El Amrani, A. & Ryan, M.D. (1999). Self-processing polyproteins - a system for co-ordinate expression of multiple proteins in transgenic plants. Plant J. 17, 453-459.

Halpin, C., Barakate, A., Askari, B., Abbott, J. & Ryan, M.D. (2001). Enabling technologies for manipulating multiple genes on complex pathways. Plant Mol. Biol.  47, 295-310.

François, I.E., De Bolle, M.F., Dwyer, G., Goderis, I.J., Woutors, P.F., Verhaert, P.D., Proost, P., Schaaper, W.M., Cammue, B.P. & Broekaert, W.F. (2002). Transgenic expression in Arabidopsis of a polyprotein construct leading to production of two different antimicrobial proteins. Plant Physiol. 128, 1346-1358.

Ma, C. & Mitra, A. (2002) Expressing multiple genes in  a single open reading frame with the 2A region of foot-and-mouth disease virus as a linker. Molecular Breeding 9, 191-199.

Ma, C & Mitra, A. (2002) Instrinsic direct repeats generate consistent post-transcriptional gene silencing in tobacco. The Plant Journal 31, 37-49.

El Amrani, A., Barakate, A., Askari, B.M., Li, X., Roberts, A.G., Ryan, M.D. & Halpin, C. (2004). Coordinate expression and independent subcellular targeting of multiple proteins from a single transgene. Plant Physiol. 135, 16-24.

Francois, I.E.J.A., Van Hemelrijck, W., Aerts, A.M., Wouters, P.F.J., Proost, .P, Broekaert, W.F. & Cammue, B.P.A. (2004). Processing in Arabidopsis thaliana of a heterologous polyprotein resulting in differential targeting of the individual plant defensins. Plant Sci. 166, 113-121.

Kwon, S.J., Hwang, E.W. & Kwon, H.B. (2004). Genetic engineering of drought resistant potato plants by co-introduction of genes encoding trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase of Zygosaccharomyces rouxii. Korean J. Genet. 26, 199-206.

Ralley, L., Enfissi, E.M.A., Misawa, N., Schuch, W., Bramley, P.M. & Fraser, P.D. (2004). Metabolic engineering of ketocarotenoid formation in higher plants. Plant J. 39 477-486.

Szymczak, A.L., Workman, C.J., Wang, Y., Vignali, K.M., Dilioglou, S., Vanin, E.F. & Vignali, D.A. (2004). Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. Nature Biotech. 22, 589-94.

Cao, Y.A., Bachmann, M.H., Beilhack, A., Yang, Y., Tanaka, M., Swijnenburg, R.J., Reeves, R., Taylor-Edwards, C., Schulz, S., Doyle, T.C., Fathman, C.G., Robbins, R.C., Herzenberg, L.A., Negrin, R.S. & Contag, C.H. (2005). Molecular imaging using labeled donor tissues reveals patterns of engraftment, rejection, and survival in transplantation. Transplantation 80, 134-139.

Szymczak, A.L. & Vignali, D.A.A. (2005). Development of 2A peptide-based strategies in the design of multicistronic vectors. Expert Opinion 5, 627-638.

Yasuda, H., Tada, Y., Hayashi, Y., Jomori, T. & Takaiwa, F. (2005). Expression of the small peptide GLP-1 in transgenic plants. Transgenic Res. 14, 677-684.

Holst, J., Szymczak-Workman, A.L., , K.M., Burton, A.R., Workman, C.J. & Vignali, D.A.A. (2006). Generation of T-cell receptor retrogenic mice. Nature Protocols 1, 406-417.

Samalova, M., Fricker, M. & Moore, I. (2006). Ratiometric fluorescence-imaging assays of plant membrane traffic using polyproteins. Traffic 7, 1701-1723.

Provost, E., Rhee, J. & Leach, S.D. (2007). Viral 2A peptides allow expression of multiple proteins from a single ORF in transgenic zebrafish embryos. Genesis 45, 625-629.

Teh, O-K & Moore, I. (2007). An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells. Nature 448, 493-497.

Wang, S., Yao, Q., Tao, J., Qiao, Y. & Zhang, Z. (2007). Co-ordinate expression of glycine betaine synthesis genes linked by the FMDV 2A region in a single open reading frame in Pichia pastoris. App. Micro. & Biotech. 77, 891-899.

Park, M., Kang, K., Park, S., Kim, Y.S., Ha, S.H., Lee, S.W., Ahn, M.J., Bae, J.M. & Back, K. (2008). Expression of serotonin derivative synthetic genes on a single self-processing polypeptide and the production of serotonin derivatives in microbes. App. Microbiol. Biotech. 81, 43-49.

Samalova, M., Fricker, M. & Moore, I. (2008). Quantitative and qualitative analysis of plant membrane traffic using fluorescent proteins. Meth. Cell Biol. 85, 353-380.

Trichas, G., Begbie, J. & Srinivas, S. (2008). Use of the viral 2A peptide for bicistronic expression in transgenic mice. BMC Biology 6, Article #40.

Ha, S.H., Liang, Y.S., Jung, H., Ahn, M.J., Suh, S.C., Kweon, S.J., Kim, D.H., Kim, Y.M. & Kim, J.K. (2010). Application of two bicistronic systems involving 2A and IRES sequences to the biosynthesis of carotenoids in rice endosperm. Plant Biotechnol J. 8, 928-938.

Mikkelsen, M.D., Olsen, C.E. & Halkier, B.A. (2010). Production of the cancer-preventive glucoraphanin in tobacco. Mol Plant. 3, 751-759.

Roongsawang, N., Promdonkoy, P., Wongwanichpokhin, M., Sornlake, W., Puseenam, A., Eurwilaichitr, L. & Tanapongpipat, S. (2010). Coexpression of fungal phytase and xylanase utilizing the cis-acting hydrolase element in Pichia pastoris. FEMS Yeast Res. 10, 909-916.

Akitake, C.M., Macurak, M., Halpern, M.E. & Goll, M.G. (2011). Transgenerational analysis of transcriptional silencing in zebrafish. Dev. Biol. 352, 191-201.

Artegiani, B., Lindemann, D. & Calegari, F. (2011). Overexpression of cdk4 and cyclinD1 triggers greater expansion of neural stem cells in the adult mouse brain. J. Exp. Med. 208, 937-948.

Bower, D.V., Sato, Y. & Lansford, R. (2011). Dynamic lineage analysis of embryonic morphogenesis using transgenic quail and 4D multispectral imaging. Genesis 49, 619-463.

Deng, W., Yang, D., Zhao, B., Ouyang, Z., Song, J., Fan, N., Liu, Z., Zhao, Y., Wu, Q., Nashun, B., Tang, J., Wu, Z., Gu, W. & Lai, L. (2011). Use of the 2A peptide for generation of multi-transgenic pigs through a single round of nuclear transfer. PLoS One 6, e19986.

Kim, J.H., Lee, S.R., Li, L.H., Park, H.J., Park, J.H., Lee, K.Y., Kim, M.K., Shin, B.A. & Choi, S.Y. (2011). High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLOS ONE 6, e18556.

Kovalskaya, N., Zhao, Y. & Hammond, R.W. (2011). Antibacterial and antifungal activity of a snakin-defensin hybrid protein expressed in tobacco and potato plants. Open Plant Sci. J. 5, 29-42.

Salguero, G., Sundarasetty, B.S., Borchers, S., Wedekind, D., Eiz-Vesper, B., Velaga, S., Jirmo, A.C., Behrens, G., Warnecke, G., Knöfel, A.K., Blasczyk, R., Mischak-Weissinger, E., Ganser, A. & Stripecke, R. (2011). Preconditioning therapy with lentiviral vector-programmed dendritic cells accelerates the homeostatic expansion of antigen-reactive human T cells in NOD.Rag1-/-.IL-2r?c-/- mice. Hum. Gene Ther. 22, 1209-1224.

Verrier, J.D., Madorsky, I., Coggin, W.E., Geesey, M., Hochman, M., Walling, E., Daroszewski, D., Eccles, K.S., Ludlow, R. & Semple-Rowland, S.L. (2011). Bicistronic lentiviruses containing a viral 2A cleavage sequence reliably co-express two proteins and restore vision to an animal model of LCA1. PLoS ONE 6, e20553.

Fang, R., Peng, Y.Q., Zheng, M. & Meng, Q.Y. (2012). Muscle-specific expression of delta-12 and omega-3 fatty acid desaturases and human catalase using "self-cleaving" 2A peptides in transgenic mice. Prog. Biochem. Biophys. 39, 175-180.

Dempsey WP, Fraser SE, Pantazis P. (2012). PhOTO zebrafish: a transgenic resource for in vivo lineage tracing during development and regeneration. PLoS One 7, e32888.

Diao, F. & White, B.H. (2012). A novel approach for directing transgene expression in drosophila: T2A-Gal4 in-frame fusion. Genetics 190, 1139-1144.

Møldrup, M.E., Geu-Flores, F., de Vos, M., Olsen, C.E., Sun, J., Jander, G. & Halkier, B.A. (2012). Engineering of benzylglucosinolate in tobacco provides proof-of-concept for dead-end trap crops genetically modified to attract Plutella xylostella (diamondback moth). Plant Biotechnol. J. 10, 435-442.

Rawlins, E.L. & Perl, A.K. (2012). The a"MAZE"ing world of lung-specific transgenic mice. Am. J. Respir. Cell. Mol. Biol. 46, 269-282.

Tittel, A.P., Heuser, C., Ohliger, C., Llanto, C., Yona, S., Hämmerling, G.J., Engel, D.R., Garbi, N. & Kurts, C. (2012). Functionally relevant neutrophilia in CD11c diphtheria toxin receptor transgenic mice. Nat. Methods 9, 385-390.

Yang, Z., Drew, D.P., Jørgensen, B., Mandel, U., Bach, S.S., Ulvskov, P., Levery, S.B., Bennett, E.P., Clausen, H. & Petersen, B.L. (2012). Engineering mammalian mucin-type O-glycosylation in plants. J. Biol. Chem. 287, 11,911-11,923.

(Top of Page)

(Return to Index)